Reference: Nadler-Holly M, et al. (2012) Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis. Proc Natl Acad Sci U S A 109(46):18833-8

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Abstract


The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Nadler-Holly M, Breker M, Gruber R, Azia A, Gymrek M, Eisenstein M, Willison KR, Schuldiner M, Horovitz A
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